The objective of the research described in this proposal is to identify new/better protein targets for vaccines, diagnostic antibodies and therapeutic antibodies against B. anthracis and Y. pestis. In addition, Diversa will apply its unique protein engineering capabilities toward isolating and optimizing diagnostic and therapeutic antibodies for better biodefense. Specific Aim 1: Characterize the proteomes of B. anthracis and Y. pestis using a proteomics approach. Specific Aim 2: Genetic analysis of proteins that are correlated with pathogen virulence. Specific Aim 3: Build a fully synthetic human antibody library & optimize a FACS-based screening approach. Using a state-of-the-art proteomics platform based upon micro 3D liquid chromatography, Diversa will identify novel proteins and measure protein expression level differences from samples of B. anthracis and Y. pestis that have been grown under various conditions, as well as coordinate (in collaboration with Dr. Arthur Friedlander (USAMRIID) and Dr. Matt Niles (U. of N. Dakota) a genetic analysis of surface and secreted proteins that are characterized in the proteomics analysis, in order to further the understanding of pathogenesis to allow better vaccines or treatments to be developed. This proposal also outlines the development of an in vitro immune system that will be independent of animal immunizations and phage display. First, Diversa will construct a synthetic human antibody library from the human genome using a proprietary process called GeneReassembly. Second, Diversa will advance high throughput screening technologies for isolating antibody leads from the library. Third, Diversa will apply its substantial expertise in protein engineering to improve antibody properties. Using its patented Gene Site Saturation Mutagenesis, Diversa will improve key properties of antibody candidates, such as binding affinity, thermal stability, solubility, and recruitment of effector functions. The development of this in vitro immune system has strong potential to create a process that generates a larger number of candidate antibodies per target and delivers candidate therapeutic antibodies to the clinic faster than current approaches. [unreadable] [unreadable] [unreadable]